CHARACTERIZATION O F YEAST CARBOXYPEPTIDASE C
Shill, J. P., and Neet, K. E. (1971), Biochem. J . 123. 283. Shill, J. P., and Neet, K . E. ( 1 974), manuscript submitted. Steitz, T . A. Fletterick, R. J., Anderson, W. F., and Bates, D. J. (1974), Biochem. SOC.Trans. 2, 52. Taylor, B. L. (1972), Ph.D. Dissertation, Case Western Re-
serve University, Cleveland, Ohio. Taylor, B. L., Barden, R. E., and Utter, M. F. (1972), J . B i d . Chem. 247, 7383. Vinograd, J., and Bruner, R. ( 1 966), Biopolymers 4 , 13 I . Wampler, D. E. (1972), Biochemistry I / , 4428.
Isolation and Partial Characterization of an Acid Carboxypeptidase from Yeast? Robert W . Kuhn,i Kenneth A. Walsh, and Hans Neurath*
ABSTRACT:
The purification and characterization of a carboxypeptidase from baker’s yeast, Saccharomyces cerevisiae, are described. The purified enzyme has been characterized with respect to molecular weight (62,000), amino acid composition, amino terminal sequence, enzymatic properties, and sensitivity to group-specific inhibitors. The enzyme is a glycoprotein composed of a single polypeptide chain, which contains 13 half-cystine residues, 2.6% amino sugars, and 12.7% neutral hexoses. The enzyme releases a variety of amino acids, includ-
ing proline, from the carboxyl terminus of proteins. Unlike pancreatic carboxypeptidases, the yeast enzyme is not inhibited by chelating agents but is inhibited by diisopropyl phosphorofluoridate, p-mercuribenzoate, mercuric chloride, N-tosyl-Lphenylalanine chloromethyl ketone, phenylglyoxal, iodoacetamide, and by photooxidation in the presence of rose bengal. The enzyme retains full activity in 4 M urea and is useful for determining carboxyl-terminal sequences of proteins and peptides.
T w o distinct classes of carboxypeptidases have been described. One class, typified by the pancreatic carboxypeptidases, exhibits a neutral to alkaline pH optimum and requires for activity divalent cations of the IIB transition series (Vallee and Riordan, 1969; Petra, 1970). The second class, termed “acid carboxypeptidases” (Zuber and Matile, 1968), displays maximal activity in the acid range and is inhibited by Dip-F.’ In contrast to the metallocarboxypeptidases, acid carboxypeptidases release from the carboxyl terminus of proteins the imino acid proline, in addition to several other amino acids. Acid carboxypeptidases have been isolated from yeast (Hata et al., 1967a,b), citrus peel (Zuber, 1964), citrus leaves (Zuber and Matile, 1968; Sprossler et al., 1971; Tschesche and Kupfer, 1972), Aspergillus (Ichishima, 1972), bean leaves (Wells, I965), cotyledons of germinating cotton seedlings (Ihle and Dure, 1972a,b), germinating barley (Visuri et al., 1969), and have been observed in a variety of plant tissues (Zuber and Matile, 1968). Cathepsin A from bovine spleen also displays several characteristics of acid carboxypeptidases (Logunov and Orekhovich, 1972).
In contrast to the extensively studied metallocarboxypeptidases, relatively little is known about the structure and mechanism of action of the acid carboxypeptidases. In order to extend our knowledge of these enzymes, we have isolated an acid carboxypeptidase from baker’s yeast. This enzyme was first described by Hata et al. (1967a) under the name of “protease C ” and certain of its chemical and enzymatic properties have been reported (Hata et al., 1967b; Aibara et al., 1971; Hayashi et al., 1970). The present paper describes a method of isolation of the enzyme and its characterization. While this work was in progress, Hayashi et al. (1973) described the isolation and some characteristic properties of this enzyme which are in close agreement with those reported herein.
From the Department of Biochemistry, University of Washington, Seattle, Washington 98195. Received April 25, 1974. Taken in part from a dissertation submitted by R. W. K u h n to the Graduate School of the University of Washington in partial fulfillment of the requirements for the degree of Doctor of Philosophy. A preliminary report has been published ( K u h n et al., 1972). This work was supported by research grants from the National Institutes of Health (GM 15731) and the American Cancer Society (BC-91). R. W. K. was supported by a training grant from the National Institutes of Health ( G M 00053) to the Department of Biochemistry. Present Address: Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77025. Abbreviations used are: Dip-F, diisopropyl phosphorofluoridate; TPCK, N-tosyl- phenylalanine chloromethyl ketone; Ac-Tyr-OEt, N-acetyl-L-tyrosine ethyl ester; Cbz-Phe-Leu, benzyloxycarboxylphenylalanylleucine; Cbz-Gly-Leu, benzyloxycarboxylglycylleucine.
* ’
PhCH2S02F, phenylmethanesulfonyl fluoride.
Experimental Section Materials Compressed baker’s yeast was obtained through the courtesy of Standard Brands, Inc. N-Acetyl- tyrosi sine ethyl ester (Ac-Tyr-OEt) was purchased from Fox Chemical Co. All carbobenzoxy dipeptides were obtained from Cyclo Chemical Corp. The B-chain of oxidized bovine insulin (lot W-I 300) was from Schwarz/Mann. DEAE-cellulose was purchased from Schleicher and Schuell, Inc. and DEAE-Sephadex A-50 and Sephadex G- 150 from Pharmacia Fine Chemicals. The protein standards used for sodium dodecyl sulfate gel electrophoresis were all obtained from Worthington Biochemical Corp. except phosphorylase b which was a gift from Dr. Edmond H . Fischer. Dip-F was purchased from Pierce Chemical Co. The reagent was diluted to 1 M in water-free 2-propanol and stored over molecular sieves (Matheson Coleman and Bell). Tetranitromethane, N-(4-dimethylamino-3,5-dinitrophenyl)maleimide, and Ellman’s reagent (5,5’-dithiobis(2-nitrobenzoic acid)) were obtained from Aldrich Chemical Co.. Inc. N BIOCHEMISTRY, VOL.
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0 Fraction
20
40
60
80 Fraction
number
F l C L R E I: Chromatography on DEAE-cellulose of the 0-90% ammonium sulfate fraction of a yeast autolysate. The column (4.0 X 60 cm) was developed at a flow rate of 200 ml/hr and 20-mI fractions were collected. Units of activity are micromoles of substrate hydrolyzed per min per ml of solution. Fractions indicated by the bar were pooled for subsequent chromatography on DEAE-Sephadex.
100
120
140
160
I80
number
F I G U R E 2: Chromatography on DEAE-Sephadex A-50 of the active enzyme fraction eluted from DEAE-cellulose (Figure 1 ). The column (2.5 X 30 cm) was developed at a flow rate of 30 ml/hr and 5-ml Fractions were collected. Units of activity are pmoles of substrate hydrolyzed per min per ml of solution. Fractions indicated by a bar were pooled for subsequent gel filtration on Sephadex G-150.
Bromosuccinimide was from Arapahoe Chemicals, Inc. Iodoadure of Weber and Osborn (1969) was used with the following cetic acid, iodoacetamide, and rose bengal were purchased modifications. Prior to electrophoresis, the protein samples from J . T. Baker Chemical Co. Mercuribenzoate was obtained were incubated for 2 hr a t 37' in 0.01 M sodium phosphate from Calbiochem. Phenylmethanesulfonyl fluoride and N (pH 7.0) containing 1% sodium dodecyl sulfate and 1% mertosyl-L.-phenylalanine chloromethyl ketone were from Cyclo captoethanol. This modification had no effect on the pattern Chemical Corp. Ethoxyformic anhydride and 1,2-epoxy-2-pheobtained when compared to gels run as suggested by Weber noxypropanewere purchased from Eastman Organic Chemiand Osborn ( I 969). Instead of the original gel buffer, a buffer cals. 1%'-Acetylimidazole and phenylglyoxal were from K & K composed of 1.45 g of NaH2P04.H20, 0.66 g of Na2HP04Laboratories, Inc. N-Ethylmaleimide was obtained from H20, and 2 g of sodium dodecyl sulfate diluted to 1 I. with disSchwarz Bioresearch Inc. Nitrophenylsulfenyl chloride was tilled water was used (private communication of M. Pangfrom Sigma Chemical Co. 2,4,6-Tribromo-4-methylcyclohexa- burn). dienone was a gift from Dr. Yigal Burstein. :V-BromoacetylAfter electrophoresis, the gels were severed a t the front of :%'-methyl-L-phenylalanine was a gift from Dr. G . M . Hass. the dye band and stained, and the mobilities were calculated by Diazo- 1 H-tetrazole was synthesized according to Sokolovsky dividing the distance of migration by the length of the gel. This and Vallee (1966). modification obviated the need for compensating for changes in Met hods gel size during staining and destaining. Amino Acid Composition. A Spinco Model 120 amino acid Carbohydrate Analysis. Total hexose content was deteranalyzer was used for all analyses. Unless otherwise noted, the mined by the phenol-sulfuric acid method of Hirs (1967). The amino acid composition was derived from triplicate analyses of results were expressed in terms of 1:1 (w/w) galactose-manprotein samples which had been hydrolyzed in 6 u HCI in nose standard, Total amino sugar was determined by the L ~ U C U Oa t 110' for 24,48, and 96 hr. Elson-Morgan method which was modified by Gatt and BerAmino Terminal Sequence Analjsis. A Beckman sequencer man (1966) using glucosamine hydrochloride as the standard. (Model 890) was used for the determination of the amino terNeuraminic acid was determined by the Warren procedure minal sequences (Edman and Begg, 1967: Herrnodson et ai., (Warren, 1959). 1972a). The phenylthiohydantoin-amino acids were identified Enzymatic Assays. Esterase activity was measured in a Raby gas chromatography with the exceptions of histidine and ardiometer TTT-1 autotitrator with 0.1 ;M N a O H as the titrant. ginine which were identified by spot tests using diazotized I Ac-'Tyr-OEt ( I O mM), dissolved in 0.01 M sodium phosphateanisidine and phenanthrenequinone, respectively. 0.15 M NaCl (pH 8.0), was routinely used as the substrate. Samples were reduced and S-carboxymethylated before seExopeptidase activity was determined with Cbz-Phe-Leu as quenator analysis as follows. The protein (5-10 mg) was disthe substrate. Hydrolysis was followed by measuring the desolved i n 5 ml of 1.0 M Tris-HCI (pH 8.0) containing 6 M crease in absorbance a t 224 nm of a 1 m v solution dissolved in guanidinium chloride. The solutin was adjusted to 0.1 M mer0.1 M sodium phosphate-0. I 5 M NaCl (pH 6.0). captoethanol by the addition of 34 p1 of the reagent ( I 5 M ) and Release of carboxyl terminal amino acids from N-substitutallowed to stand a t room temperature under nitrogen for 4 hr. ed dipeptides was recorded continuously utilizing a Technicon The solution was then adjusted to 0.1 1 M iodoacetate by the autoanalyzer essentially as described by Lenard et ai. (1965). addition of 1.2 ml of 0.5 M reagent. After 20 min, 34 pI of 15 Chart speed was set at 1 in./min. Portions of the reaction mixM mercaptoethanol was added and the solution dialyzed in a ture were continuously withdrawn, a t the rate of 0.33 ml/min. rocking, flow-through dialysis apparatus against 20 1. of 5% through Teflon tubing which passed through a boiling water acetic acid for 36 hr. All manipulations following the addition bath and then into the analytical system of the analyzer. Subof iodoacetate were performed in the dark. After dialysis the strates (0.25-10 mM) were dissolved in 0.05 M 2-(iV-morpholiprotein sample was lyophilized, suspended in 33% acetic acid, no)ethanesulfonic acid-0.1 5 M NaCl (pH 6.0) containing 10% and introduced into the reaction vessel of the sequencer. dioxane. The method was calibrated with standard solutions of Sodium Dodecyl Sulfate Gel Electrophoresis. The procethe appropriate amino acids.
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CHARACTERIZATION OF YEAST CARBOXYPEPTIDASE C
-__ -
.-**.e
_-
TABLE I:
Purification of Yeast Protease C
Step Autolysis a t pH 7 . 0 Autolysis a t pH 5 . 0 90zammonium sulfate DEAE-cellulose DEAE-Sephadex SeDhadexG-150 FIGURE 3: Gel filtration on Sephadex G-150 of the p l e d fractions from DEAE-Sephadex chromatography (Figure 2). The column size was 2.5 X 120 cm. Fraction volume was 5 ml. The p l e d fractions were precipitated by the addition of sufficient solid ammonium sulfate to reach 90% saturation. The precipitate was dissolved in 5 ml of elution buffer (0.07 M sodium phosphate (pH 7.0) containing 0.15 M NaCI). A flow rate of 25 ml/hr was maintained by the hydrostatic pressure of a 40-cm head. Right insert: analytical disc gel electrophoresis of protease C. Gels are from left to right: gel electrophoresis in the presence of mercaptaethanol and sodium dadecyl sulfate: pH 8.3 system containing urea: standard pH 8.3 system. Migration was from top to bottom (cathode to anode). Gels were severed at the dye front before staining. Left insert: Schlieren optical pattern of yeast protease C after 75-min centrifugation at 60,Ohl rpm. The protein concentration was 6.0mg/ml, the bar angle 60°. Sedimentation was from right to left.
Heat Inactivation. Samples (0.5 ml) of a solution of protease C (0.1 mg/ml in 0.01 M sodium phosphate-0.15 M NaCl (pH 7.0)) were placed in test tubes which were sealed with parafilm. The tubes were placed in a constant-temperature bath a t the desired temperature and, a t appropriate times, removed and cooled in an ice bath, and peptidase activity was determined. Effects of Urea. The enzyme was dissolved in 0.1 M sodium phosphate-0.15 M NaCl (pH 6.5) containing a predetermined concentration of urea. After incubation for 30 min a t 25' peptidase activity was determined using substrate with or without urea. p H Stability. Enzyme ( 1 mg/ml) was dissolved in buffers of 0.1 M sodium acetate (pH 3.4, or 5 ) . sodium phosphate (pH 6 or 7). or sodium borate (pH 8 . 9 or 10) which had been adjusted to ionic strength 0.1 5 M. A t zero time and after 2 hr aliquots were assayed for peptidase activity. Results
Isolation and Purification of Yeas1 Protease C. Yeast protease C was purified by a modification of the procedure of Hata et a / . (1967b). Compressed baker's yeast (24 Ibs) was crumbled and mixed with 5 I. of chloroform. The mixture was occasionally stirred for 3 hr during which time the yeast became liquified. Excess chloroform was removed by aspiration, 6 I. of distilled water was added, and the p H was adjusted to 7.0 by the dropwise addition of 1.0 N N a O H . After the mixture was allowed to autolyze overnight a t room temperature with constant stirring, cellular debris was removed by centrifugation. The supernatant (10.7 I.) contained no detectable esterase activity. The enzyme was activated by adjusting the supernatant to pH 5.0 by the dropwise addition of 1.0 N HCI and allowing the protein to undergo autolysis overnight a t room temperature. The fully active autolysate was brought to pH 7.0 by the dropwise addition of 1.0 N N a O H and then to 90% satura-
Vol (ml)
Total Protein (mg)
Total Units
Specific Yield Activity (Z)
10,650 238,560
0
10,650 213,596
37,275
0.175 100
.1,092 17,080
37,128
2.17
99.6
732.8 18,320 353.2 16,560 233.2 13,986
25.0 47
49.1 44.4 37.5
1,145 184 585
0
60
tion by the addition of solid ammonium sulfate while the pH was maintained at 7.0 with 1.0 N N a O H . The precipitate which formed overnight (at 4' with stirring) was collected by centrifugation a t 20,OOOg for 30 min, dissolved in a minimal amount of water. and dialyzed against two changes of distilled water and finally against 0.01 M sodium phosphate buffer (pH 7.0) containing 0.15 M NaCI. The dialyzed solution was applied to a DEAE-cellulose column (4.0 X 100 cm) which was previously equilibrated with 0.01 M sodium phosphate buffer (pH 7.0) and then developed by applying a linear gradient of NaCl (0.15-0.45 M) in the same buffer (Figure I ) . Active fractions were pooled and the protein was precipitated by the addition of sufficient solid ammonium sulfate to reach 90% saturation. The precipitate was collected by centrifugation for 30 min at 20,OOOg dissolved in a minimal amount of sodium phosphate buffer containing 0.1 5 M NaCI, and dialyzed overnight against the same buffer. The dialyzed solution was applied to a column (2.5 X 50 cm) of DEAE-Sephadex A-50 which was previously equilibrated with 0.01 M sodium phosphate buffer (pH 7.0) containing 0.15 M NaCI. The column was developed by applying a linear gradient of NaCl (0.15-0.60 M) in the same buffer (Figure 2). Active fractions were pooled and the protein was precipitated with ammonium sulfate as before. The precipitate was dissolved in a minimal amount of phosphate buffer containing 0.15 M NaCl and dialyzed overnight against the same buffer. The solution was applied to a column (2.5 X 120 cm) of Sephadex (3-150 which was previously equilibrated with 0.01 M sodium phosphate buffer (pH 7.0) containing 0.15 M NaCI. Fractions eluted from the column were tested for activity with the results shown in Figure 3. Fractions with constant specific activity were p l e d , dialyzed against three changes of 6 1. of distilled water, and lyophilized. The solid protein was stored at -2OO. The purification and yields of the enzyme are summarized in Table 1. Physicochemical Characterization of Yeast Protease C. The homogeneity of the enzyme prepared in this manner was demonstrated by electrophoresis on polyacrylamide gels at in pH 8.3 in the presence of 8 M urea: a single narrow band was observed (Figure 3). The diffuse band in the absence of urea may be due to self-association of the enzyme. Supporting evidence for homogeneity was obtained by sedimentation in the ultracentrifuge. A sample of the lyophilized protein was dissolved and dialyzed against a potassium phosphate buffer (pH 6.5) of 0.1 ionic strength and examined in the ultracentrifuge (Figure 3). The symmetrical nature of the boundary (0.6% protein) . ~4.6 S). suggests a high degree of homogeneity ( ~ 2 0 = BIOCHEMISTRY. VOL.
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TABLE 11 : Amino
Acid Composition of Protease C.
Amino Acid
LYS Pro Thr
Residues/Molecule"
T Yr
Aspartic acid Threonine * Serine' Glutamic acid Proline Glycine Alanine Valined Met hioninee Isoleucined Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan/ Cystine
68.5 19.5 31.1 45.3 25.8 36.2 24.0 31.6 6.6 19.7 35.0 23.2 23.5 16.7 8.0 8.8 12.1 13,' 12.6€
Phe
0
40
80
120
I60 200 2 4 0
Time
(minutes)
280 3 2 0
F I G U R E 5 : Time course of the release of amino acid residues from the B-chain of oxidized bovine insulin by yeast protease C. Protein concentration was 4 mg/ml in 0.1 K pyridine-acetate, pH 6.0. The reaction was performed at 25" with a substrate to enzyme ratio of 300. The results are in agreement with the actual sequence of -Phe-Tyr-Thr-ProLys- Ala-OH.
Average values of nine analyses based o n 24.0 mol of alanine,'62,000. Extrapolated t o zero time of hydrolysis. Determined as Cm-cysteine after reduction and alkylation. Values after 96-hr hydrolysis. e Half-cystine a n d methionine were determined as cysteic acid and methionine sulfone, respectively, after performic acid oxidation. Determined by the method of Edelhoch (1967). Additional evidence for the homogeneity of protease C was obtained by polyacrylamide gel electrophoresis of the protein in the presence of sodium dodecyl sulfate and mercaptoethanol. The gel shown in Figure 3 revealed a single band corresponding in mobility to a molecular weight of 62,000. Chemical purity was established by sequenator analysis which gave lysine as the sole amino-terminal residue. An extended analysis revealed the following amino terminal sequence' L j s- Ile- Lys-Asp- Pro-Lys- Ile-Leu-GIy- Ile- Asp-Pro. The amino acid composition of yeast protease C is given in Table II.The value reported for half-cystine was obtained after reduction and carboxymethylation ( 13.6 residues/molecule). Performic acid oxidation yielded 12.6 residues of cysteic acid
per molecule. In the absence of denaturing agents, the protein failed to react with the Ellman reagent (Ellman, 1959) but in the presence of 8 M urea, 1. I residue of thiol per protein molecule reacted (P. M. Levy, unpublished observations). These results agree with those reported by Hayashi et al. (1973). The protein was found to be a glycoprotein containing 2.6% amino sugars and 12.7% neutral hexose, but no sialic acid. Measurements of the stability showed that the enzyme was stable for 2 h r in the pH range 5.0-8.0 at 25' and between pH 5.0 and 7.0 at 37'. The enzyme was completely active when exposed for 5 min to 55' at pH 7.0; 50% of the activity was lost after 5 min at 62" while at 68' under these conditions, all activity was lost. A plot of In k l (where k , is the first-order rate constant of inactivation) vs. 1/T was linear, the slope corresponding to a value of 73 kcal/mol for the heat of inactivation. Concentrations of urea up to 4 M had no effect upon enzyme activity while at higher concentrations activit) was rapidly lost. Preincubation in urea followed by assay in the absence of urea consistently yielded higher enzyme activity than when the enzyme was assayed in the presence of urea, suggesting that urea denaturation was reversible. Enzymatic Properties. The pH dependence of esterase and peptidase activities is shown in Figure 4. Each activity reveals a -~~ ~~
~
Hydrolysis by Yeast Protease C of N-Substituted L-Dipeptides of the Type Cbz-X-Leu.
TABLE 111: -1.4
- 12
6 ?
Substrate
D
pH dependence of peptidase and esterase activities of yeast protease C. Peptidase activity is expressed as per cent of the maximum rate of hydrolysis of Cbz-Phe-Leu. The results of esterase hydrolysis (Ac-Tyr-OEt) are expressed as k,,,. Above p H 9.5, ester hydrolysis could not be determined due to base-catalyzed hydrolysis of the substrate. F I C L R E 4:
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Cbz-Gly -Leu Cbz-Phe-Leu Cbz-Ala-Leu Cbz-Val-Leu Cbz-Leu-Leu Cbz-Ile-Leu Cbz-Ser-Leu Cbz-His-Leu Cbz-Pr o -Le u Cbz-Glu-Leu Cbz-Nle-Leu
kc, t (min-')
364 905 21,723 848 2,568 90 1 1,075 84 49 1,792 2,620
K,,, (mbi) 2 0 1 0 0 0 1
95 24 28 10
12 10 7
1 8
1 8 8 3 0 03
k,,t/Km 123 3,709 21,658 8,230 22,137 8,743 632 47 27 216 43,667
Excess Substrate Inhibition
+-
+ + + -
+
CHARACTERIZATION
OF YEAST CARBOXYPEPTIDASE C
Hydrolysis by Yeast Protease C of N-Substituted Dipeptides Of the Type Cbz-Gly-X.
TABLE I V :
Cbz-Gly -Leu Cbz-Gly -Phe Cbz-Gly-phe Cbz-Gly -Arg Cbz-Gly -Glu Cbz-Gly -Gly Acetyl-Gly-Leu
364 135
2.95 123 1.2 116 N o t hydrolyzed 40 16.3 2.4 40 24.1 1.7 Hydrolyzed but kinetics constants not measurable 115 45.3 2.5
relatively sharp pH optimum with an apparent maximum at pH 6.0 for peptidase activity and at pH 7.5 for esterase activity . The specificity of the enzyme was determined using the oxidized B chain of insulin as a substrate. Figure 5 shows that the enzyme released free amino acids from the carboxyl terminus in a sequential manner. I n addition to aromatic and neutral amino acids, lysine and proline were released in nearly stoichiometric amounts. The ability of protease C to release acidic amino acids at pH 4.15 was previously demonstrated in the sequence analysis of monkey amyloid protein A (Hermodson et a/., 1972b). The action of the enzyme on a series of N-substituted dipeptides reveals that at least three sites of the substrate influence the hydrolysis rates. An analysis of the hydrolysis of a series of N-carbobenzoxy dipeptides of the type Cbz-X-Leu, in terms of TABLE
the kinetic parameters k,,, and K,, is shown in Table I l l . If the penultimate amino acid is an aromatic or a long chain hydrophobic residue, K , is decreased by one order of magnitude as compared to substrates containing in this position alanyl, glycycl, or hydrophilic residues. Substrates containing more hydrophobic side chains can bind in a manner producing excess substrate inhibition. I f one of the hydrogen atoms of glycine is replaced by a methyl group, a marked enhancement of K,,, is observed while K , is essentially unchanged (compare Cbz-GlyLeu with Cbz-Ala-Leu). Substitution of alanine by valine or leucine results in a decrease in both k,,, and K,. I f the penultimate amino acid is histidine or proline, k,,, is decreased by one order of magnitude as compared to Cbz-Gly-Leu while K , is unchanged. The lowest K , is observed for the peptide containing norleucine as the penultimate amino acid. Variation of the carboxyl terminal amino acid also affects the kinetic constants (Table IV). I f Cbz-Gly-Leu is used as the standard of comparison, substitution of either arginine (basic) or glutamic acid (acidic) for leucine reduces k,,, by one order of magnitude and increases K,. The substitution of the aromatic residue phenylalanine for leucine affects neither parameter significantly. Comparison of the rate of hydrolysis of Cbz-Gly-Leu with that of acetyl-Gly-Leu (Table I V ) shows that varying the blocking group affects K , more than k,,,. Effect of Inhibitors. The ability of a number of different group-specific reagents to inhibit yeast protease C is documented in Table V. Typical sulfhydryl reagents, including HgC12, p-mercuribenzoate, and iodoacetamide, inhibit the enzyme but their reaction with residues other than cysteine (Webb, 1966) has not been excluded in this work. Reaction
v: Effect of Inhibitors on Yeast Protease C.’ Inhibitor
Probable Site of Action
Condition
Method
I. Inhibition p-Mercuribenzoate HgCL 1,2-Epoxy-3-phenoxypropane Dip-F PhCHzS02F TPCK Phenylglyoxal Photooxidation Iodoacetamide Ethoxyformic anhydride 11. N o Inhibition Iodoacetic Nitrophenylsulfonyl chloride N-Bromosuccinimide 2,4,6-Tribromo-4-methylcyclo hexadienone N-Bromoacetyl-N-methyl+ phenylalanine Tetranitromethane N-Acetylimidazole Diazo-1 H-tetrazole N-Ethylmaleimide N-(4-Dimethylamino-3,5dinitropheny1)maleimide
Cys, His Cys, His Met, Glu, Asp Ser Ser His Arg Cys, Trp, His, Met, Tyr Cys, His, Lys His, Glu, Asp, Lys, Tyr
1 mMb 1 mMb 0.1 MC 1 mMb 1 rnM’ 2 mMb Rose Bengal dye’ 0 . 2 MC 100-fold molar excess
Boyer (1954) Webb (1966) Tang (1971) Hartley (1960) Gold (1967) Schoellman and Shaw (1963) Takahashi (1968) Abe et a/. (1971) Gurd (1 967) Huc et al. (1971)
Cys, His, Lys TrP Trp, Met, Cys TrP
0 . 2 Me 100-fold molar excesse 100-fold molar excesse 100-fold molar excesse
Gurd (1967) Scoffone et al. (1968) Spande and Witkop (1967) Burstein and Patchornik (1972)
Cys, His, Lys, Tyr, -COO+, Met, NH, TYr Tyr, His, Lys, Cys Tyr, His CYS CYS
1.5Zd
Hass and Neurath (1971a) 50-fold molar excess 50-fold molar excess 50-fold molar excess 1 mMb 1 mM’
Sokolovsky et a/. (1966) Simpson et a / . (1963) Sokolovsky and Vallee (1 966) Riordan and Vallee (1967) Witter and Tuppy (1960)
a Enzyme concentration, 1 mg/ml. 0.1 M sodium phosphate-0.15 M NaCl (pH 7.0). 0.1 M sodium phosphate-0.15 M NaCl (pH 6.5). 0.1 M sodium phosphate-0.15 M NaCl ( p H 7.5). e 0.25 M sodium acetate J p H 4.5). .’0.02 M sodium veronal-0.5 M NaCl (pH 7.5). 0.25 M sodium acetate.
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with typical tyrosine reagents fails to inactivate the enzyme even when up to five tyrosine residues per molecule are modified (tetranitromethane). Tryptophan-directed reagents ( N bromosuccinamide) also failed to cause inhibition after modification of three residues. The presence of carboxyl groups near the active site is suggested by the transient inhibition by ethoxyformic anhydride (Huc et al., 1971), by the inhibition by 1,2-epoxy-3-phenoxypropane(Tang,197 1). and by the observation that the neutral alkylating agent iodoacetamide inhibits the enzyme while iodoacetate has no effect. Yet, N-bromoacetyl-lY-methyl-L-phenylalanine,which reacts specifically with G I u z ~of~ bovine carboxypeptidase A (Hass and Neurath. 197 1 b) and B (Hass et al., 1972), has no effect on yeast protease C. The presence of an active serine in the enzyme is indicated by the inhibition by Dip-F and phenylmethanesulfonyl fluoride. Total loss of activity is observed coincident with the incorporation of 0.98 mol of Dip-F/mol of enzyme, in agreement with the recent report by Hayashi et al. (1973). A serine residue is likewise the site of phosphorylation ( R . W. Kuhn and H . Neurath, i n preparation). The enzyme is also inhibited by N-tosyl-L-phenylalanine chloromethyl ketone, by photooxidation in the presence of rose bengal and by phenylglyoxal ( R . W. Kuhn and H. Neurath. in preparation). I n contrast to the pancreatic carboxypeptidases, yeast protease C is not inhibited by the metal-chelating agents EDTA and I , IO-phenanthroline. Discussion The present procedure of isolating yeast protease C represents an improvement over that of Hayashi et al. (1973) for operation at a laboratory scale: reduction of solution volumes occurs a t early stages, chromatography on DEAE-cellulose is more effective because of gradient rather than stepwise elution. and one DEAE-Sephadex column is eliminated. A double peak is routinely observed on DEAE-Sephadex (Figure 2). and attempts to distinguish between the two forms with regard to amino terminal sequence, amino acid composition, molecular weight, and kinetic parameters have been unsuccessful. Rcchromatography on DEAE-Sephadex A-50 following dialysis and lyophilization yields only one peak of protein and enzyme activity. The enzyme isolated by this procedure is free of protease A. an endopeptidase which can easily contaminate the product. and appears to be homogeneous by several criteria. The enzyme contains an amino-terminal lysine residue in agreement with the observation of Aibara et al. ( I 97 I ). Yeast protease C releases a variety of free amino acids from proteins and synthetic 1%’-carbobenzoxy dipeptides. Experiments with these dipeptides indicate that at least three sites of the substrate affect hydrolysis rates. Substrates containing charged amino acids in the carboxyl-terminal position yield higher K,, and lower k,;,, values than those with hydrophobic residues. The type of residue in the penultimate position also is a major determinant of both K , and kc:!,.Substitution of alanine for glycine i n the penultimate position results in a marked increase in k,,,. The effect upon K , produced by varying the nature of the blocking group suggests that this site of the substrate also influences hydrolysis rates. Experiments with the oxidized B chain of bovine insulin indicate that the enzyme sequentially releases free amino acids from the carboxyl-terminus. Particularly noteworthy is the release of proline. These experiments also revealed that the enzyme preparation was free of any contaminating endopeptidase activity. Although no homology could be detected between the amino terminal sequence of yeast protease C and any other protease.
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the enzyme does share common catalytic features with both the metallocarboxypeptidases and the serine proteases. Whereas the enzyme is not inhibited by chelating agents, and thus is not a metalloenzyme, the inhibition by phenylglyoxal implicates the presence of an arginine a t or near the active site. This residue may be involved in binding the carboxyl group of peptide substrates as in the case of bovine carboxypeptidase A (Lipscomb et al., 1969). The catalytic mechanism has features in common with the serine proteases. The inhibition by Dip-F and phenylmethanesulfonyl fluoride indicates the presence of an active serine, while inhibition by N-tosyl-I_-phenylalanine chloromethyl ketone implicates a histidine residue at the active site. Aibara et a / . (1971) have found that a free sulfhydryl group is required for enzyme activity. This interpretation is based largely on the inhibition by p-mercuribenzoate and by HgCI2 and on reaction wjith 5,5’-dithiobis(2-nitrobenzoicacid) and is consistent with the present observations of inhibition by several sulfnydryl reagents. The stability of the enzyme in urea solutions and its ability to release a wide variety of carboxyl-terminal amino acids from proteins and peptides at both neutral and acidic pH values render it highly useful for sequence determination (Hermodson r t a/., 1972b). Acknowledgments The authors are indebted to Dr. G . Michael Hass and I>r. Yigal Burstein for their advice, to L. H . Ericsson for the sequenator analyses, and to Richard R. Granberg for the amino acid analyses. References
19. 1 9 7 4
Abe, T., Takahashi, K., and Ando, T. (1971), J . Biochem. 6 9 . 363. Aibara. S . , Hayashi, R., and Hata, T. (1971), Agr. Biol. C‘hem. 35,658. Boyer, P. D. ( I 954), J . Amer. Chem. soc. 76, 433 1 , Burstein. Y.. and Patchornik. A. (1972). Biochemisrrj, 1 1 , 4641. Edelhoch, H . ( 1 967), Biochemistry 6, 1948. Edman, P., and Begg. G. ( 1 967), Eur. J . Biochem. I . 80. Ellman, G . L. (1959). Arch. Biochem. Biophys. 82, 70. Gatt, S., and Berman, E. R . (l966), Anal. Biochem. I S . 167. Gold, A. M . ( 1967). Methods Enzymol. I I . 706. Curd. F. R. N . (1967). Methods Enzymol. 1 1 , 532. Hartley, B. S. ( I 960), Annu. Rev. Biochem. 29, 45. Hass, G . M.,Covier, M. A,. Grahn, D. T.. and Neurath, H. ( I 972), Biochemistrj. 1 1 , 3787. Hass. G . .V.,and Neurath, H. (1971a), Biochemistry IO. 3535. Hass, G. M., and Neurath, H . ( 1 97 1 b), Biochemistrj. 10, 3541. Hata. T., Hayashi. R.. and Doi, E. ( I 967a), Agr. Biol. C‘hem. 31. 150. Hata, T., Hayashi. R.. and Doi, E. (1967b), Agr. B i d . C‘hrtti. 31. 357. Hayashi, R., Aibara. S., and Hata, T. (1970). Biochirn. Biophys. Acta 212, 359. Hayashi, R., Moore, S., and Stein, W. H . (1973), J . Biol. Chem. 248, 2296. Hermodson, M. A , , Ericsson, L. H.. Titani. K., heurath, H.. and Walsh, K. A. ( I 972a), Biochrmistrj 11. 4493. Hermodson, M. A,, Kuhn, R . W., Walsh, K. A.. Neurath. H.. Eriksen. N.. and Benditt, E. P. (1972b). Biochemistrj, 11. 2934. Hirs. C. H. W. ( I967), Methods Enzymol. I I , 44 I . Huc, C., Olomucki, A,. Lan, L.. Phy, B.. and Van Thoui, W . (1971). Eur. J . Biochem. 21. 161.
REACTION O F PYRIDOXAL SULFATE WITH
AN AMINOTRANSFERASE
Ichishima, E. ( 1 972), Biochim. Biophys. Acta 258, 274. Ihle, J. W., and Dure, L. S., 111 (1972a), J . Biol. Chem. 247, 5034. Ihle, J. W., and Dure, L. S., 111 (1972b), J . Biol. Chem. 247, 5041. Kuhn, R. W., Walsh, K. A., and Neurath, H. (1972), Fed. Proc., Fed. Amer. Soc. Exp. Biol. 31, 877. Lenard, J., Johnson, S. L., Hyman, W., and Hess, G. P. ( 1 965), Anal. Biochem. I 1, 30. Lipscomb, W. W., Hartsuck, J. A., Reeke, G. W., Jr., Quiocho, F. A., Bethge, P. H., Ludwig, M. L., Steitz, T. A,, Muirhead, H., and Copolla, J. C. (1969), Brookhaven Symp. Biol. 21, 24. Logunov, A. I . , and Orekhovich, V. W. (1972), Biochem. Biophys. Res. Commun. 46, 116. Petra, P. H. (1970), Methods Enzymol. 19, 460. Riordan, J. F., and Vallee, B. L. (1967), Methods Enzymol. 11. 541. Schoellman, G., and Shaw, E. (1963), Biochemistry 2, 252. Scoffone, E., Fontana, H., and Rocchi, R. (1968), Biochemistry 7, 971. Simpson, R. T., Riordan, J. F., and Vallee, B. L. (1963), Biochemistry 2, 6 16.
Sokolovsky, M., Riordan, J. F., and Vallee, B. L. ( I 966), Biochemistry 5. 3582. Sokolovsky, M., and Vallee, B. L. (1966), Biochemistry, 5, 3574. Spande, T. F.,and Witkop, B. (1967), Methods Enzymol. 1 1 , 498. Sprossler, B . , Heilman, H. D., Grumpp, E., and Uhlig, H. (1971), Hoppe-Seyler’s Z . Physiol. Chem. 352, 1524. Takahashi, K. (1968),J. Biol. Chem. 243,6171. Tang, J. (1971),J. Biol. Chem. 246, 4510. Tschesche, H., and Kupfer, S. (1972), Eur. J . Biochem. 26, 33. Vallee, B. L., and Riordan, J. R. ( I 969), Brookhaven Symp. Biol. 21, 91. Visuri, K., Mikola, J., and Enori, T.-M. (1969), Eur. J. Biorhem. 7, 193. Warren, L. ( 1 959), J . Biol. Chem. 234, 1971. Webb, J. L. (1966), in Enzyme and Metabolic Inhibitors, Vol. 2, New York, N. Y., Academic Press, p 729. Weber, K., and Osborn, M. ( 1 969), J . Biol. Chem. 244,4406. Wells, J. R. E. ( 1 965), Biochem. J . 97, 228. Witter, A., and Tuppy, H. ( I 960), Biochim. Biophys. Acta 45, 429. Zuber, H. (1964), Nature (London) 201, 613. Zuber, H., and Matile, Ph. ( 1 968), Z . Naturforsch. B 23, 663.
Reaction of Pyridoxal 5’-Sulfate with Apoenzyme of Aspartate Aminotransferase. Covalent Labeling of the Protein with Elimination of Sulfate? In-yu Yang,’ Radii M. Khomutov,§ and David E. Metzler*
ABSTRACT: The 5’-sulfate esters of pyridoxine, pyridoxal, and pyridoxamine have been prepared and their absorption spectra and dissociation constants have been determined. Spectra have been resolved using log normal distribution functions. The pK, values are compared with those of related compounds. That for the pyridinium group is strikingly lower in pyridoxal sulfate than in pyridoxal phosphate. When pyridoxal sulfate reacts a t pH 8.3 with apoaspartate aminotransferase it slowly forms an absorption band at 24.9 k K (402 nm) which is narrow and displays marked vibronic fine structure. (This is in sharp contrast to the broader band at 27.4 k K [364 nm] formed with pyridoxal phosphate.) At lower pH the peak shifted to 25.9 k K (386 nm) and a t higher pH to 27.4 kK. The pyridoxal sulfate-enzyme form absorbing at 24.9 k K has positive circular dichroism (like the native enzyme). Upon denaturation with acid
the chromophore precipitated with the protein suggesting covalent attachment. Cysteine, /3-mercaptoethylamine, ethylenediamine, ethanolamine, serine, and arginine all react with pyridoxal sulfate with gradual production of narrow absorption bands similar to that formed with the apoenzyme. Investigation of the reaction with cysteine reveals that inorganic sulfate is eliminated during the reaction at a rate that is identical with that of formation of the 24.9-kK chromophore. Investigation with nmr shows that formation of a thiazolidine ring is followed by loss of the 4’ proton and disappearance of the 5’-CH2 peak. Elimination of sulfate to a quinonoid form that tautomerizes to a cyclic substituted Schiff base is proposed. I t is speculated that an unknown group X in the enzyme replaces the S H group of cysteine in a similar reaction.
o n e approach to understanding the function of the phosphate group in the coenzyme pyridoxal 5’-phosphate (pyri-
doxal-P)’ is to study analogs of similar geometry and charge type but containing different anionic side chains. Thus, replacement of the phosphorylated hydroxymethyl side chain of pyridoxal-P with a propionic acid side chain (5-deoxy-5’-carboxymethylpyridoxal) yields an analog of the coenzyme that binds to several apoenzymes. However, the activity with apo aspartate aminotransferase (apoAAT) is a t most 3% of that of
From the Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50010. Received April 29, 1974. This study was supported by Grant AM-01549 from the National Institutes of Health, U. S. Public Health Service. Present address: Department of Biochemistry, University of California, Berkeley, Calif. 94720. Present address: Institute of Molecular Biology, Academy of Sciences of the USSR. Moscow. USSR I I73 12.
I Abbreviations used are: AAT, aspartate aminotransferase; pyridoxal-P, pyridoxal 5’-phosphate.
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